Project summary Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Endoscopic surveillance with biopsy is the standard method for early CRC diagnosis and mortality reduction. However, conventional endoscopic screening is limited by incomplete bowel cleansing, limited morphological detail in visible light observations of the mucosal surface, and lack of molecular specificity of the visible findings. New imaging approaches with sensitive and specific biomarkers capable of differentiating CRC lesions from healthy and inflammatory non-tumor sites are urgently needed for CRC early detection. The objective of this proposal is to validate a new family of CRC-targeting probes for fluorescence colonoscopy and SPECT imaging using a small peptide with the amino-acid sequence c[CTPSPFSHC]OH (TCP-1) identified from mouse CRC using phage display. Our preliminary studies have shown that TCP-1 can specifically recognizes human CRC cells and tumor vasculature, but not inflammatory tissues. The primary hypothesis to be tested is that fluorescent and radiolabeled TCP-1 peptide analogues have a fundamentally distinct targeting capability with high CRC selectivity. We have proposed two specific aims to test our hypothesis: 1) Synthesize and optimize TCP-1 probes. To bring CRC targeting probes to a clinically translatable level, we will incorporate an optimal radionuclide 99mTc and near infrared fluorescence (NIRF) cyanine 7 (Cy7) dye into the targeting probes using different chelators and inserting spacers between the peptide and chelate to obtain the most promising fluorescence and 99mTc- labeled TCP-1 imaging agents, respectively, for studies in Aim 2. Serial in vitro cell binding, blocking, and internalization studies will be performed in human HT29 and HCT116 CRC cells using four 99mTc-TCP-1 analogues, two Cy7-TCP-1 candidates, and their negative control peptides to evaluate their cell binding specificity and affinity, as well as biodistribution and kinetic profiles in healthy mice. 2) Characterize the in vivo targeting properties of the most promising TCP-1 probes for detection of CRC lesions. Using a dual-mode (fluorescence and white light) murine endoscopy and a high-resolution SPECT imager, one Cy7-TCP-1 and one 99mTc-TCP-1 most promising probe from Aim 1 will be evaluated to validate their CRC targeting capabilities in two orthotopic mouse CRC models in inflamed colon and non-inflamed colon that involves transplantation of xenografted and primary human CRC tissue onto the cecum. Specificity of the TCP-1 probe for CRC targeting will be determined by in vivo blocking studies. Ex vivo assessments, including biodistribution, autoradiograph analysis, fluorescence examination and pathological identification will reveal the variability of TCP-1 probe uptake in CRC and inflammatory tissues. Successful outcome of this proposal may ultimately enable CRC patients to undergo a complementary diagnostic approach for precision cancer management.